M A I - L O R Y .4ND TRAVIS
Human Pancreatic Enzymes: Purification and Characterization of a Nonelastolytic Enzyme, Protease E, Resembling Elastaset Peter A. Mallory and James Travis*
ABST RACl : An enzyme with proteolytic activity has been isolated froin activated extracts of human pancreatic tissue. The purification procedure included salt fractionation followed by ion-exchange chromatography on SE-Sephadex C-25 and on DEAE-Sephadex A-50. The homogeneity of this enzyme, designated protease E, was demonatrated by disc electrophoresis and by sedimentation equilibrium centrifugation studies. The homogeneous enzyme shows the ability to hydrolyze many of the conventional synthetic substrates used for the identification of elastase activity; however, i t demonstrates no significant elastolytic activity. A comparison of human protease E with porcine elastase reveals a high degree of similarity between the two proteases
with respect to inhibition by active-site directed peptide chloromethyl ketones, stability, decreased susceptibility to naturally occurring proteinase inhibitors, and specificity for synthetic substrates as well as several other physical properties. The major difference between human protease E and porcine elastase, other than the lack of elastolytic activitl by human protease E, seems to be in the ionic character and the amino acid composition of these two proteins. Porcine elastase is a cationic enzyme, while human protease E appears to be anionic in nature. These dissimilarities concerning elastolytic activity and ionic character appear to be directly related.
E l a s t a s e , by definition, is a proteolytic enzyme which has the ability to digest elastin, as well as other protein substrates. This elastin-digesting capability sets it apart from other pancreatic endopeptidases in that it is the only enzyme with such a function. Since the initial report by Balo and Banga (1950) concerning the elastolytic activity of porcine pancreatic tissue, a wealth of iiiformation has been obtained on porcine elastase (Mandl, 1962; Shotton, 1970; Hartley and Shotton, 1971). Conflicting reports as to whether an elastolytic enzyme is secreted from the human pancreas have been made over the past several years (Hall et ai., 1952; Lamy and Tauber, 1,963: Trowbridge and Moon, 1969). However, Trowbridge and Moon (1972) reported that a purified preparation of human pancreatic elastase had been obtained by salt fractionation of homogenized pancreatic tissue followed by adsorption of the enzyme on powdered human elastin 1%tailed characterization of this molecule, however, was not accomplished. Clemente et ai. (1972), employing imniunological techniques and synthetic substrates, observed the presence of two protein components with elastase-like esterase activity in activated human pancreatic juice. Feinstein er al. (1974) have recently reported the partial purification and characterization of‘ two human pancreatic enzymes which demonstrate Ac(Ala)30fvLfe’esterase activity. They have termed these two enzymes “elastases.” In our laboratory, using activated extracts of human pancreatic tissue, we have also been able to detect two enzyme components which hydrolyze A ~ ( A l a ) ~ 0 M One e . of these components, however, has no appreciable elastolytic activi-
ty. It does nevertheless have many striking properties in common with porcine pancreatic elastase. The purification and characterization of this enzyme, which we have called human protease E, are reported in this communication.
+ IIroni the Department of Biochemistry, Ilniversit\ of Georgi>i. Athens. Georgia 30601. Received August 2Y, 1974. Supported i n part by National Institutes of Health Grant HL-13778 and by a grant from the Tobacco Research Council. One of u s ( J . T ) is a Career Dwelupiiient Awardee of the National Institutes of Health.
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kxperimental Section
Maleriais Human pancreas were obtained quick frozen at autopsy from St. Joseph’s Hospital, Marshfield, Wis., and Athens General Hospital, Athens, Ga. Pipes and elastin-orcein were purchased from Calbiochem. Cyclo Chemical Company provided CAP and TBAP. Powdered bovine neck ligament elastin, Congo Red-elastin, Tos-Lys-CHzC1, and TosPhe-CH2CI were products of Sigma Chemical Company. Chicken ovomucoid, soybean trypsin inhibitor, lima bean trypsin inhibitor, Kunitz bovine pancreatic trypsin inhibitor, and chromatographically purified porcine elastase were obtained from Worthington Biochemical Company. iPr2FP was a product of Aldrich Chemical Company. DEAE-Sephadex A-50 as well as SE-Sephadex C-25 were from Pharmacia Fine Chemicals. Ac(Ala)jOMe was obtained from Miles Research Laboratories. Hippuryl-I--arginine and hippuryl-l.-phenylalanine were purchased from Mann Research Laboratories. Bz-Leu-OEt was a gift of Dr. .I. Folk, __ Abbreviations used are: iPr2FP. diisopropyl phosphorofluoridate: Tos-l.ys-CH2C1, l-chloro-3-tosylarnido-7-aminoheptanone; Tos-PheClilCl, I -I-to~qlaniido-2-phenylethqlchloromethyl ketone; Pipes, pipsr;lrine-.l,.l”-bis(2-ethanesulfonicacid) monosodium nionohydr:ite: RrArgOEt. .~‘-beriioyl-l--arginineethql ester: R ~ l e u O E t ben~oql-I , lcucine ethyl ester; TusArgOMe, toluenesulfonyl-I -arginine inethyl ester: Suc(Ala)jNA, succinyl-~-alanyl-~.-alanyl-~.-al~inir~e p - riitrc~diiilide; /tc(Ala),OMe. ncet)l-t.-alanyl-l -alanyl-l.-alaninc incth)l ester: 1-Ala-NP. carboxybenzoyl- alanine p-nitrophenjl ester; r - Boc- AlaYP. terr-butLloxycarbon)I-L-alanine p-nitrophenyl eater: AcTyrOEt. ;icet>I-1 -tyrosine ethyl ester. __.________.__I_--
i
HUMAN PANCREATIC ENZYMES, PROTEASE E
pH4.6
\L
pH 6.5
3
FRACTION
NUMBER
F I G U R E 1 : SE-Sephadex C-25 chromatography of activated acetone powder extracts of human pancreatic tissue. The column was equilibrated with 0.005 M Pipes-HCI (pH 6.5) containing 0.025 M CaC12. The column was alternately washed with 0.005 M acetic acid-0.025 M CaClz (pH 4.6) and 0.005 M Pipes-HCI-0.025 M CaC12 (pH 6.5) as indicated. Column dimensions, 1.7 X 28 cm; flow rate, 20 ml/hr; fraction size, 5 ml. Curves are designated as follows: optical density at 280 n m ( 0 - - O ) , left ordinate, activity against BzArgOEt, (@-a). right ordinate; activity against Z-AlaN P (x- - - -x), right ordinate.
National Institutes of Health, Bethesda, Md., and Suc(Ala)3-NA was a gift of Dr. J. Bieth, Civil Hospital, Strasbourg, France. Porcine pancreatic secretory trypsin inhibitor was a gift of Dr. L. J . Greene, Brookhaven National Laboratory, Upton, N.Y. The Bowman-Birk soybean trypsin inhibitor was donated by Dr. I. E. Liener, University of Minnesota, St. Paul, Minn. The five active-site specific inhibitors of porcine elastase were generously provided by Dr. J. C. Powers, Georgia Institute of Technology, Atlanta, Ga. Homogeneous a-1-antitrypsin was prepared in our laboratory (Travis and Pannell, 1973). All other reagents were of analytical grade obtained from various commercial sources.
Methods Enzyme Assays. Elastase esterolytic activity was measured spectrophotometrically at room temperature using Z-Ala-NP or t-Boc-Ala-NP in 0.05 M Pipes (pH 6.5) (Visser and Blout, 1972). One unit of activity was defined as an absorbance change of one optical density unit per minute at 347.5 nm. Specific activity was calculated as units of esterase activity per milligram of protein. Ac(Ala)@Me and Suc-(Ala)3-NA were assayed by the method of Bieth and Meyers (1973) and Bieth et a f . (1974), respectively. Protein concentration was determined spectrophotometrically by the method of Warburg and Christian ( I 942). For purified preparations of enzyme a specific extinction coefficient of 24 5 ( 1 % solution, 280 nm), as determined in this paper, was utilized. Proteolytic activity was measured by the casein hydrolysis method of Kunitz as described by Laskowski ( 1 9 5 5 ) . Elastin digestion assays were routinely performed by the method of Gertler and Birk (1970). lnhibition Studies. Inhibition experiments using naturally occurring proteinase inhibitors were carried out by mix-
ing various quantities of inhibitor with enzyme in 0.05 M Tris-HC1 (pH 8.0) containing 0.05 M CaC12. After incubation for 45 min at 25O, the mixtures were assayed for proteolytic activity as described above. The enzyme concentration used was M. Inhibition by peptide chloromethyl ketones was determined spectrophotometrically with t-Boc-Ala-NP. The appropriate inhibitor was dissolved in 0.1 M Pipes (pH 6.5) containing 10% (v/v) methanol prior to incubation with enzyme at 25'. Aliquots of the incubation mixture were removed at regular intervals for assay. The concentrations of inhibitor and enzyme were and IOd5 M, respectively. Polyacrylamide Electrophoresis. Acid and alkaline disc electrophoresis were carried out by the procedure of Brewer and Ashworth (1969). The gels were 7.5% acrylamide with a running pH of 2.3 and 8.3, respectively. Sodium Dodecyl Sulfate Gel Electrophoresis. Protein solutions were electrophoresed in 10% acrylamide gels after incubation in sodium dodecyl sulfate solutions as described by Weber and Osborn (1969). The molecular weight of human protease E was determined using proteins of known molecular weights as standards. Gel Chromatography. The estimation of the molecular weight of human protease E was performed by chromatography 011 Sephadex G-100 as described by Whitaker (1963). Standard proteins of known molecular weight were used for comparisons. Analytical Ultrucentrifugation and Amino Acid Analysis. Experiments to determine amino acid composition and analytical ultracentrifuge studies to determine sedimentation coefficient, molecular weight, and extinction coefficient of the protease preparation were carried out as previously described (Mallory and Travis, 1973). For ultracenBIOCHEMISTRY, VOL.
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!d
'D
.5
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F I G L J R E 2:
DEAE-Sephadex A-50 chromatography of active fractions from SE-Sephadex C-25 column chromatography. The column was equilibrated with 0.005 M Pipes-HCI (pH 6.5), containing 0.025 M CaC12, and eluted with a linear gradient to 0.125 M CaCI: as indicated. Column dimensions, 1.7 X 26 cm; flow rate, 20 ml/hr: fraction size, 5 ml. Curves are designated as follows: optical density a t 280 nm (O---O), left ordinate; activity against BzArgOEt ( 0 - O ) , right ordinate; activity against 2 - A l a - N P ( w - - - - w ) , right ordinate.
trifugation experiments requiring high protein concentrations, iPrzFP human protease E was utilized (see Stability). Results
Purification of Human Protease E . In initial attempts to isolate the anionic trypsin component in activated human pancreatic extracts (Mallory and Travis, 1973), it became apparent that the anionic fraction containing the trypsin activitj was also composed of a second enzyme possessing strong proteolytic activity. This protease was insensitive to both Tos-Lys-CHzCI and Tos-Phe-CH2Cl but was rapidly inhibited by iPrlFP (see Inhibition Studies). Because this second protease had strong elastase esterase activity, it was felt that the purification and characterization of the molecule would be of significant interest. Unless otherwise stated, all operations were performed a t 4', and aqueous solutions were prepared in double distilled water. IYI T l A L PROCEDURF. Human protease E purification followed the experimental conditions for the isolation of
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human anionic trypsin (Mallory and Travis, 1973). Briefly, 40 g of acetone powder, representing 1000 g of pancreatic tissue, was extracted in 0.01 M HCI (pH 2.6), salt fractionated between 0.2 and 0.8 with solid ammonium sulfate, and d i a l y ~ e da t alkaline pH against 0.05 M Tris-HC1 (pH 8.0) containing 0.05 M CaC12, to remove salt and initiate activation of zymogens. The details of this procedure have been reported previously (Coan et ai., 1971). SE-SEPHADEXc-2s c H R O M A T O G R A P H Y . The activated material obtained after dialysis was adjusted to pH 4.6 with 0.1 M acetic acid, diluted with water to an ionic strength equivalent to that of a buffer consisting of 0.005 M Pipes-0.025 M CaClz (pH 6 . 5 ) (buffer A), and applied to a column of SE-Sephadex C-25 equilibrated in the same buffer. The column was then washed with 0.005 M acetate buffer (pH 4.5) containing 0.025 M CaC12, until the A280 was less than 0.020. When the column was developed by addition of buffer A, a single protein peak was eluted containing approximately 30-40% of the applied CAP esterase activity (Figure 1) as well as trypsin esterase activity due to anionic
H U M A N PANCREATIC ENZYMES, PROTEASE E
Table I: Purification of Protease E from Human Pancreas.* Total Protein Fractionation Step 1. Crude extract 2. 0.2-0.8 salt fraction 3. SE-Sephadex C-25 column 4. DEAE-Sephadex A-50 column
(mg)
4000 1360 81 36
Total Activity (units x 20Ob 18@ 150’ 78
(%)
Specific Activity (units/mg)
100
0.05
93 a1 39
0.14
2.8
1.85
37.0 520.0
Recovery
Purification
Elastolytic ActivityC
1.o
26.00
___
0.83 7.80 0.01
Purification based on human protease E esterase activity toward Z-Ala-NP, assayed as described under Methods. b Corrected for nonspecific esterase activity due t o trypsin and chymotrypsin. e Elastolytic activity measured on elastin Congo Red as described under Methods. T h e activity of pure porcine elastase was taken as 100, after 1-hr digest. d Milligrams of 24.5. protein determined using Ezs0 “(,l%) -
trypsin. The significant loss of esterase units was foum !o be primarily due to the removal of large amounts of cationic trypsin and chymotrypsin, both of which show considerable 2-Ala-NP esterase activity (Visser and Blout, 1972; Janoff, 1969). DEAE-SEPHADEXA-50 CHROMATOGRAPHY. The peak tubes of the SE-Sephadex chromatography were pooled and applied to a DEAE-Sephadex A-50 column equilibrated against buffer A. After the passage of inactive unretarded protein, human protease E was eluted by initiation of a linear gradient from 0.025 M CaClz to 0.125 M CaC12, both in 0.005 M Pipes buffer (pH 6.5). As shown in Figure 2, human protease E eluted separately from anionic trypsin a t about 0.1 M CaC12 concentration. The purification scheme is shown in Table 1. Because of the instability of anionic trypsin, any delay following SE-Sephadex chromatography resulted in substantially lower amounts of this enzyme whereas the recovery of human protease E was not noticeably affected. Criteria of Homogeneity and Molecular Weight Studies. Human protease E routinely eluted from DEAE-Sephadex A-50 with constant specific activity. Rechromatography of peak fractions on DEAE-Sephadex resulted in a single peak with constant specific activity identical with the applied material. Additional evidence of the homogeneity of this preparation was obtained by subjecting it to gel filtration on Sephadex (3-75. The preparation eluted as a single symmetrical peak with a constant specific activity comparable to that of the applied enzyme. Analytical disc electrophoresis of human protease E in 7.5% gels a t p H 2.3 and 8.3 showed only a single band with no minor contaminants, again indicative of homogeneity (Figure 3). Sedimentation velocity experiments were performed on iPr2FP enzyme in 0.005 M Pipes-0.05 M CaC12 (pH 6.5) a t protein concentrations of IO and 7.5 mg/ml; both resulted in singly symmetrical Schlieren peaks and a sedimentation constant ( ~ 2 0 . ~of) 3.19 S was calculated from these data. Boundary depletion sedimentation equilibrium experiments were used to calculate the molecular weight of iPr2FP human protease E. A protein concentration of 0.19 mg/ml in 0.005 M Pipes-0.025 M CaC12 (pH 6.5) was used with a rotor speed of 40,000 rpm. The molecular weight calculated from these data was 31,059, using a partial specific volume of 0.722 calculated from the amino acid composiof 24.5 was tion. An extinction coefficient (El cm(l%)) computed from ultracentrifuge studies using interference optics (Babul and Stellwagen, 1969). The extinction coefficient experiments were conducted in 0.005 M Pipes-0,025
FIGURE 3: Polyacryiamme ansc eiectropnoresis ot human protease E (80 pg). Patterns were stained with 1% Amido Schware in 7.5% acetic acid (A) pH 2.3, 7.5% gel, direction of migration is from anode (top) 10 cathode (bottom): ( B ) pH 8.3, 7.5%. direction of migration is from cathode (tap) to anode (bottom).
C a C h (pH 6.5) using iPr2FP enzyme concentrations of 1.7 and 2.7 mg/ml. Sodium dodecyl sulfate gel electrophoresis of iPr2FP human protease E was performed after incubation in sodium dodecyl sulfate solution in order to determine both homogeneity and molecular weight. A single component wa* detected and a molecular weight of 30,500 was estimated using proteins of known molecular weights as standards. The molecular weight of human protease E as estimated by gel filtration chromatography on Sephadex G-100 was 30.000. Amino Acid Composition. The amino acid composition of human protease E is presented in Table 11. These data were calculated from analysis after 22-, 48-, and 72-hr hydrolysis of I-mg samples of enzyme. The number of residues of each amino acid present was based on a n assumed value of 20 residues of alanine per molecule of protein. A molecular weight of 30,792 was calculated from these data. The amino acid compositions of porcine elastase (Shotton, 1970) and the human “elastase 1” isolated by Fzinstein et al. (1974) are also presented in Table I1 for comparison. Stability. Human protease E was quite stable in dilute solutions of protein concentrations less than 0.4 mg/ml within a pH range of 4.0-8.0. No loss of activity could be M
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_..
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-.
-_ -
Table 11: Amino Acid Compositiona of Human Protease E and Other Related Proteases. Human Protease E
Other Related P r o t e a s e s ____
Time of Hydrolysis -
Amino Acid
22 h r
48 hr
72 hi-
Value Taken
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half -Cystine \'aline Me thi iiriiiie Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
8.2 6.7 10.9 28.5 16.1 23.9 24.2 18.3 31.3
9 .o 8.1 9.6 27.4 15.4 23.8 25.1 15.2 35.9
11.I 7.4 10.0 29.4 14.9 23.6 24.1 15.8 38.4
22.1
22.9
24.9
12.4 20.1 8.5 7.6
12.9 20.6 7.6 7.1
13.3 20.9 7.3 6.4
9.4 7.4 10.0 28.4 16.4' 24.Zb 24.4 17.4 35.2 20.0 15.3' 25.3' 0 .9' 13.7' 20.5 7 .a 7.1 9 .Od N o . of residues Mol wt
Near e s t Integer 9 7 10 28 16 24 24 17 35 20 15 25
1 14 21
8 7 9 290 30.792
P o r c me Elastase'
Human "E last as e l>>f
3 8 6 6 12 10 24 25 19 14 22 16 19 21 7 16 25 26 17 16 8 16 27 24 2 0 10 10 18 17 11 7 3 6 7 ____ -1.1 .d 240 238 25.900 25.050
________ _ - __ . . ~ Data are expressed as amino acid residues/molecule, assuming 20 alanines/molecule of protein. b Threonine and serine values are based on linear extrapolations to zero time, and valine and isoleucine values are based on linear extrapolation t o 96 hr. Averages of 22-hr hydrolysates of oxidized samples. Tryptophan determined by average of 22-hr hydrolysates in the presence of 4 % thioglycolate. e Shotton (1970). f Feinstein e t al. (1974). -
~
a
Table 111: Inhibition of' Human Protease E" with Active-Site Specific Peptide Chloromethyl Ketone Inhibitors of Porcine Pancreatic Elastase. - __~ _ _ _ _ ~ ~ ~ _ _ _ _ _ _ . ._ ~ _ _ _ _ Relative Reactivity'
_
Inhibitor 0.7 7 1.o 1.o Ac-Ala-Ala-AlaCH,Cl 6.5 10 7.5 75 10.7 1.6 Z -Gly - Leu-AlaCH,Cl 6.5 10 20.4 204 34.6 18 Ac - Ala- Ala- Ala- AlaC H,C 1 6.5 10 6.5 10 61 610 88 71 Ac -Ala- Ala- P r o - AlaCH,Cl 13.8 138 19.7 16 Ac -Ala- Ala- Phe - AlaCH,C 1 6.5 10 _______ __ .a Human protease E concentration: 10-5 M, all enzyme assays were performed using t-Boc-Ala-NP as described under Methods. Kinetic constants calculated by the method of Powers and Tuhy (1973). Averages of at least four runs. Controls containing no inhibitor exhibited no significant decrease in esterolytic activity. Relative reactivity assuming Ac-Ala-AlaAlaCH2C1 is equal to 1.0. These values for relative comparison only (see Discussion). These constants were taken from Powers and Tuhy (1973) and were calculated by Powers and Tuhy (1973) for p H 6.5 from original inhibition studies a t p H 5.0. ~
detected after 3 months, even at 24'. Below p H 4.0, however, the enzyme gradually lost activity, presumably due to irreversible denaturation. At protein concentrations above 0.4 mg/ml, rapid autolysis occurred; therefore, for experiments requiring high protein concentrations, iPrzFP enzyme was employed. The pH optimum for protease E was determined on casein substrates using the following buffers: 0.1 M sodium citrate (pH 2.0-5.5), 0.1 M Pipes-HCI (pH 6.0-7.7), 0.1 21 Tris-HCI (pH 7.2-8.0), and 0.1 M sodium carbonate (pH 7.7- 10.5). The pH optimum for casein hydrolysis was be-
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tween pH 7.7 and p H 9.5. Effect of Inhibitors on Human Protease E. S Y ~ T I H I ; . T I < ' I N H I B I T O R S . In order to determine the degree of similarity between porcine elastase and human protease E, several synthetic active-site directed reagents designed specifically to inhibit porcine elastase were tested (Powers and Tuhy. 1973). The data were processed using a least-squarcs computer program and the results are listed in Table 111. All of the compounds tested were found to be strong inhibitors of human protease E a t p H 6.5. NATURALLYO C C U R R I N G INHIBITORS. Human prOte-
_
HUMAN
PANCREATIC ENZYMES,
PROTEASE E
Table IV: Inhibition Spectrum of Human Protease Ea (Expressed as % Proteolytic Activity Remaining).
Table V: Relative Proteolytic Activitya of Several Human and Porcine Pancreatic Proteases. Relative Activity
Molar Ratio Inhibitor : Enzyme Porcine Inhibitor Soybean trypsin Lima bean trypsin Kunitz bovine pancreatic trypsin Chicken ovomucoid porcine pancreatic secretory trypsin Bowman-Birk (soybean) Human s e r u m a,-antitrypsin
1:l 83 96 60 100 95 96 0
5:l 64 91 55 96 91 94
1O:l 59 85 39 89 85 86
Assays for inhibition of proteolytic activity were performed at p H 8.0 as described under Methods. a
ase E was also tested against a wide range of naturally occurring inhibitors including soybean trypsin inhibitor, lima bean trypsin inhibitor, Kunitz bovine pancreatic trypsin inhibitor, chicken ovomucoid, porcine Kazal inhibitor, Bowman-Birk (soybean) trypsin inhibitor, and a-l-antitrypsin. With the notable exception of a-1-antitrypsin, all the naturally occurring inhibitors tested showed a surprising lack of inhibition even at inhibitor-enzyme molar ratios of 1O:l. These results are summarized in Table IV. Although detailed results have not been published, porcine elastase has reportedly little if any susceptibility to chicken ovomucoid (Mandl, 1962), pancreatic trypsin inhibitor (Mandl, 1962), or the Bowman-Birk soybean inhibitor (Gertier and Birk, 1970). Human serum a t 1:lOO dilutions, however, has been found to inhibit porcine elastase from 50 to 90%, while human protease E (0.1 mg/ml) is totally inhibited by a 1: 100 dilution of human serum. In particular, human protease E and porcine pancreatic elastase have both been shown to be subject to complete inhibition by a-1-antitrypsin even a t 1: 1 molar ratios of inhibitor to enzyme. Substrate Specificity and Kinetic Parameters. Human protease E was tested against three synthetic substrates which are commonly used to measure elastase activity. The human enzyme was found to have strong activity toward Z-Ala-NP and t-Boc-Ala-NP. The specific activity of human protease E toward the former was 25.8 as compared to 26.9 for porcine elastase. The K, value for the latter substrate using human protease E was determined from eight different substrate concentrations (ranging from 2.0Km to O.lK,)) to be 1.66 X M. The K , value for this substrate using porcine elastase was reported by Visser and Blout (1972) to be 3.0 X l o w 4 M . Ac(Ala)3OMe, a highly specific substrate for porcine elastase (Gertler and Hofmann, 1970), was also hydrolyzed by human protease E but a t only 25% the rate of that for porcine elastase. Human protease E was also tested against Suc(Ala)3NA, another highly specific substrate for porcine elastase (Bieth et al., 1974). Although the substrate was hydrolyzed, the rate was only 7.5% of that of porcine elastase. This was, however, 500 times greater than the rate of substrate hydrolysis by porcine chymotrypsin and 1000 times greater than hydrolysis by porcine trypsin (Bieth et al., 1974). Human protease E showed no detectable activity toward BzArgOEt or TosArgOMe, both synthetic substrates for trypsin or toward AcTyrOEt or BzLeuOEt which a r e used to detect human and bovine chymotrypsin and bovine chy-
Protein Substrate
Elas-
Casein Undyed elastinb Elastin-Orceinb Elastin Congo Red
100 100
tase
100 100
Human Porcine Protease Trypsin E 98 1.50 0.66 0.17
0.50 0.28 0.10
Human Trypsin 108
0.70 0.04 0.06
a Porcine elastase has been assigned a value of 100. Assay procedure as described by Shotton (1970). Values obtained from 8-hr incubation of protease with substrate.
motrypsin C activity, respectively. In addition, no carboxypeptidase A or B activity could be measured using hippurylL-phenylalanine and hippuryl-L-arginine as substrates. Strong proteolytic activity against casein and hemoglobin was observed with human protease E representing 98% of that shown by porcine elastase when equal milligram quantities of both proteases were tested on these same substrates. Assays f o r Elastolytic Activity. Using the procedure as described by Shotton (1 970), Congo Red-elastin, orceinelastin, and powdered bovine elastin were used to assay for elastolytic activity. Although a strong elastolytic activity was detected in the initial steps of the purification procedure, the protein which we have isolated has no significant elastolytic activity. In addition to the above procedures, the elastin-plate assay as described by Sbarra et a f . (1960) also yielded negative results. The adsorption of human protease E on elastin was measured by the method of Gertler (1971). Using an assay mixture containing 0.125 mg of human protease E in 4.0 ml of 0.1 M borate buffer (pH 8.8), we found that less than 1% of the C A P esterase activity of human protease E was adsorbed on 20 mg of elastin. In identical experiments with porcine elastase, 96% of the Z-Ala-NP esterase activity was adsorbed on elastin and complete digestion occurred within 2 hr. In order to ascertain the absolute elastolytic activity of human protease E as related to other pancreatic proteases, equal microgram quantities of porcine elastase, porcine trypsin, human protease E, and human trypsin were tested against a variety of elastin substrates. The results of this experiment are shown in Table V. Although human protease E does demonstrate trace activity against the elastin substrates tested, this activity would appear to be insufficient to justify calling this enzyme an elastase. Discussion Previous communications regarding the properties of human pancreatic proteolytic enzymes have suggested that there are two elastases present in activated extracts of human pancreatic tissue and in activated human pancreatic juice (Feinstein et al., 1974; Clemente et al., 1972). In our laboratory, however, we have been able to detect only one protein component which possesses elastolytic activity. Yet, we have resolved two proteases which show strong elastaselike esterase activity. The more anionic of these components we have called human protease E. Although this enzyme does have many properties in common with the well-characBIOCHEMISTRY, VOL.
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Table VI: Comparative Properties of Human Protease E and Porcine Elastase. ~~~___________
Property
Human Protease E
Porcine Elastase@
3.19 S
2.60 S
31,059 30,792
25,000 25,900
Extinction coefficient
24.5
23.6
( E j c m(lrY>l) pH optimum I
Human Pancreatic Enzymes: Purification and Characterization of a Nonelastolytic Enzyme, Protease E, Resembling Elastaset Peter A. Mallory and James Travis*
ABST RACl : An enzyme with proteolytic activity has been isolated froin activated extracts of human pancreatic tissue. The purification procedure included salt fractionation followed by ion-exchange chromatography on SE-Sephadex C-25 and on DEAE-Sephadex A-50. The homogeneity of this enzyme, designated protease E, was demonatrated by disc electrophoresis and by sedimentation equilibrium centrifugation studies. The homogeneous enzyme shows the ability to hydrolyze many of the conventional synthetic substrates used for the identification of elastase activity; however, i t demonstrates no significant elastolytic activity. A comparison of human protease E with porcine elastase reveals a high degree of similarity between the two proteases
with respect to inhibition by active-site directed peptide chloromethyl ketones, stability, decreased susceptibility to naturally occurring proteinase inhibitors, and specificity for synthetic substrates as well as several other physical properties. The major difference between human protease E and porcine elastase, other than the lack of elastolytic activitl by human protease E, seems to be in the ionic character and the amino acid composition of these two proteins. Porcine elastase is a cationic enzyme, while human protease E appears to be anionic in nature. These dissimilarities concerning elastolytic activity and ionic character appear to be directly related.
E l a s t a s e , by definition, is a proteolytic enzyme which has the ability to digest elastin, as well as other protein substrates. This elastin-digesting capability sets it apart from other pancreatic endopeptidases in that it is the only enzyme with such a function. Since the initial report by Balo and Banga (1950) concerning the elastolytic activity of porcine pancreatic tissue, a wealth of iiiformation has been obtained on porcine elastase (Mandl, 1962; Shotton, 1970; Hartley and Shotton, 1971). Conflicting reports as to whether an elastolytic enzyme is secreted from the human pancreas have been made over the past several years (Hall et ai., 1952; Lamy and Tauber, 1,963: Trowbridge and Moon, 1969). However, Trowbridge and Moon (1972) reported that a purified preparation of human pancreatic elastase had been obtained by salt fractionation of homogenized pancreatic tissue followed by adsorption of the enzyme on powdered human elastin 1%tailed characterization of this molecule, however, was not accomplished. Clemente et ai. (1972), employing imniunological techniques and synthetic substrates, observed the presence of two protein components with elastase-like esterase activity in activated human pancreatic juice. Feinstein er al. (1974) have recently reported the partial purification and characterization of‘ two human pancreatic enzymes which demonstrate Ac(Ala)30fvLfe’esterase activity. They have termed these two enzymes “elastases.” In our laboratory, using activated extracts of human pancreatic tissue, we have also been able to detect two enzyme components which hydrolyze A ~ ( A l a ) ~ 0 M One e . of these components, however, has no appreciable elastolytic activi-
ty. It does nevertheless have many striking properties in common with porcine pancreatic elastase. The purification and characterization of this enzyme, which we have called human protease E, are reported in this communication.
+ IIroni the Department of Biochemistry, Ilniversit\ of Georgi>i. Athens. Georgia 30601. Received August 2Y, 1974. Supported i n part by National Institutes of Health Grant HL-13778 and by a grant from the Tobacco Research Council. One of u s ( J . T ) is a Career Dwelupiiient Awardee of the National Institutes of Health.
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kxperimental Section
Maleriais Human pancreas were obtained quick frozen at autopsy from St. Joseph’s Hospital, Marshfield, Wis., and Athens General Hospital, Athens, Ga. Pipes and elastin-orcein were purchased from Calbiochem. Cyclo Chemical Company provided CAP and TBAP. Powdered bovine neck ligament elastin, Congo Red-elastin, Tos-Lys-CHzC1, and TosPhe-CH2CI were products of Sigma Chemical Company. Chicken ovomucoid, soybean trypsin inhibitor, lima bean trypsin inhibitor, Kunitz bovine pancreatic trypsin inhibitor, and chromatographically purified porcine elastase were obtained from Worthington Biochemical Company. iPr2FP was a product of Aldrich Chemical Company. DEAE-Sephadex A-50 as well as SE-Sephadex C-25 were from Pharmacia Fine Chemicals. Ac(Ala)jOMe was obtained from Miles Research Laboratories. Hippuryl-I--arginine and hippuryl-l.-phenylalanine were purchased from Mann Research Laboratories. Bz-Leu-OEt was a gift of Dr. .I. Folk, __ Abbreviations used are: iPr2FP. diisopropyl phosphorofluoridate: Tos-l.ys-CH2C1, l-chloro-3-tosylarnido-7-aminoheptanone; Tos-PheClilCl, I -I-to~qlaniido-2-phenylethqlchloromethyl ketone; Pipes, pipsr;lrine-.l,.l”-bis(2-ethanesulfonicacid) monosodium nionohydr:ite: RrArgOEt. .~‘-beriioyl-l--arginineethql ester: R ~ l e u O E t ben~oql-I , lcucine ethyl ester; TusArgOMe, toluenesulfonyl-I -arginine inethyl ester: Suc(Ala)jNA, succinyl-~-alanyl-~.-alanyl-~.-al~inir~e p - riitrc~diiilide; /tc(Ala),OMe. ncet)l-t.-alanyl-l -alanyl-l.-alaninc incth)l ester: 1-Ala-NP. carboxybenzoyl- alanine p-nitrophenjl ester; r - Boc- AlaYP. terr-butLloxycarbon)I-L-alanine p-nitrophenyl eater: AcTyrOEt. ;icet>I-1 -tyrosine ethyl ester. __.________.__I_--
i
HUMAN PANCREATIC ENZYMES, PROTEASE E
pH4.6
\L
pH 6.5
3
FRACTION
NUMBER
F I G U R E 1 : SE-Sephadex C-25 chromatography of activated acetone powder extracts of human pancreatic tissue. The column was equilibrated with 0.005 M Pipes-HCI (pH 6.5) containing 0.025 M CaC12. The column was alternately washed with 0.005 M acetic acid-0.025 M CaClz (pH 4.6) and 0.005 M Pipes-HCI-0.025 M CaC12 (pH 6.5) as indicated. Column dimensions, 1.7 X 28 cm; flow rate, 20 ml/hr; fraction size, 5 ml. Curves are designated as follows: optical density at 280 n m ( 0 - - O ) , left ordinate, activity against BzArgOEt, (@-a). right ordinate; activity against Z-AlaN P (x- - - -x), right ordinate.
National Institutes of Health, Bethesda, Md., and Suc(Ala)3-NA was a gift of Dr. J. Bieth, Civil Hospital, Strasbourg, France. Porcine pancreatic secretory trypsin inhibitor was a gift of Dr. L. J . Greene, Brookhaven National Laboratory, Upton, N.Y. The Bowman-Birk soybean trypsin inhibitor was donated by Dr. I. E. Liener, University of Minnesota, St. Paul, Minn. The five active-site specific inhibitors of porcine elastase were generously provided by Dr. J. C. Powers, Georgia Institute of Technology, Atlanta, Ga. Homogeneous a-1-antitrypsin was prepared in our laboratory (Travis and Pannell, 1973). All other reagents were of analytical grade obtained from various commercial sources.
Methods Enzyme Assays. Elastase esterolytic activity was measured spectrophotometrically at room temperature using Z-Ala-NP or t-Boc-Ala-NP in 0.05 M Pipes (pH 6.5) (Visser and Blout, 1972). One unit of activity was defined as an absorbance change of one optical density unit per minute at 347.5 nm. Specific activity was calculated as units of esterase activity per milligram of protein. Ac(Ala)@Me and Suc-(Ala)3-NA were assayed by the method of Bieth and Meyers (1973) and Bieth et a f . (1974), respectively. Protein concentration was determined spectrophotometrically by the method of Warburg and Christian ( I 942). For purified preparations of enzyme a specific extinction coefficient of 24 5 ( 1 % solution, 280 nm), as determined in this paper, was utilized. Proteolytic activity was measured by the casein hydrolysis method of Kunitz as described by Laskowski ( 1 9 5 5 ) . Elastin digestion assays were routinely performed by the method of Gertler and Birk (1970). lnhibition Studies. Inhibition experiments using naturally occurring proteinase inhibitors were carried out by mix-
ing various quantities of inhibitor with enzyme in 0.05 M Tris-HC1 (pH 8.0) containing 0.05 M CaC12. After incubation for 45 min at 25O, the mixtures were assayed for proteolytic activity as described above. The enzyme concentration used was M. Inhibition by peptide chloromethyl ketones was determined spectrophotometrically with t-Boc-Ala-NP. The appropriate inhibitor was dissolved in 0.1 M Pipes (pH 6.5) containing 10% (v/v) methanol prior to incubation with enzyme at 25'. Aliquots of the incubation mixture were removed at regular intervals for assay. The concentrations of inhibitor and enzyme were and IOd5 M, respectively. Polyacrylamide Electrophoresis. Acid and alkaline disc electrophoresis were carried out by the procedure of Brewer and Ashworth (1969). The gels were 7.5% acrylamide with a running pH of 2.3 and 8.3, respectively. Sodium Dodecyl Sulfate Gel Electrophoresis. Protein solutions were electrophoresed in 10% acrylamide gels after incubation in sodium dodecyl sulfate solutions as described by Weber and Osborn (1969). The molecular weight of human protease E was determined using proteins of known molecular weights as standards. Gel Chromatography. The estimation of the molecular weight of human protease E was performed by chromatography 011 Sephadex G-100 as described by Whitaker (1963). Standard proteins of known molecular weight were used for comparisons. Analytical Ultrucentrifugation and Amino Acid Analysis. Experiments to determine amino acid composition and analytical ultracentrifuge studies to determine sedimentation coefficient, molecular weight, and extinction coefficient of the protease preparation were carried out as previously described (Mallory and Travis, 1973). For ultracenBIOCHEMISTRY, VOL.
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!d
'D
.5
Fln Z
13
,O
1.5
FRACTION
NUMBER
F I G L J R E 2:
DEAE-Sephadex A-50 chromatography of active fractions from SE-Sephadex C-25 column chromatography. The column was equilibrated with 0.005 M Pipes-HCI (pH 6.5), containing 0.025 M CaC12, and eluted with a linear gradient to 0.125 M CaCI: as indicated. Column dimensions, 1.7 X 26 cm; flow rate, 20 ml/hr: fraction size, 5 ml. Curves are designated as follows: optical density a t 280 nm (O---O), left ordinate; activity against BzArgOEt ( 0 - O ) , right ordinate; activity against 2 - A l a - N P ( w - - - - w ) , right ordinate.
trifugation experiments requiring high protein concentrations, iPrzFP human protease E was utilized (see Stability). Results
Purification of Human Protease E . In initial attempts to isolate the anionic trypsin component in activated human pancreatic extracts (Mallory and Travis, 1973), it became apparent that the anionic fraction containing the trypsin activitj was also composed of a second enzyme possessing strong proteolytic activity. This protease was insensitive to both Tos-Lys-CHzCI and Tos-Phe-CH2Cl but was rapidly inhibited by iPrlFP (see Inhibition Studies). Because this second protease had strong elastase esterase activity, it was felt that the purification and characterization of the molecule would be of significant interest. Unless otherwise stated, all operations were performed a t 4', and aqueous solutions were prepared in double distilled water. IYI T l A L PROCEDURF. Human protease E purification followed the experimental conditions for the isolation of
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human anionic trypsin (Mallory and Travis, 1973). Briefly, 40 g of acetone powder, representing 1000 g of pancreatic tissue, was extracted in 0.01 M HCI (pH 2.6), salt fractionated between 0.2 and 0.8 with solid ammonium sulfate, and d i a l y ~ e da t alkaline pH against 0.05 M Tris-HC1 (pH 8.0) containing 0.05 M CaC12, to remove salt and initiate activation of zymogens. The details of this procedure have been reported previously (Coan et ai., 1971). SE-SEPHADEXc-2s c H R O M A T O G R A P H Y . The activated material obtained after dialysis was adjusted to pH 4.6 with 0.1 M acetic acid, diluted with water to an ionic strength equivalent to that of a buffer consisting of 0.005 M Pipes-0.025 M CaClz (pH 6 . 5 ) (buffer A), and applied to a column of SE-Sephadex C-25 equilibrated in the same buffer. The column was then washed with 0.005 M acetate buffer (pH 4.5) containing 0.025 M CaC12, until the A280 was less than 0.020. When the column was developed by addition of buffer A, a single protein peak was eluted containing approximately 30-40% of the applied CAP esterase activity (Figure 1) as well as trypsin esterase activity due to anionic
H U M A N PANCREATIC ENZYMES, PROTEASE E
Table I: Purification of Protease E from Human Pancreas.* Total Protein Fractionation Step 1. Crude extract 2. 0.2-0.8 salt fraction 3. SE-Sephadex C-25 column 4. DEAE-Sephadex A-50 column
(mg)
4000 1360 81 36
Total Activity (units x 20Ob 18@ 150’ 78
(%)
Specific Activity (units/mg)
100
0.05
93 a1 39
0.14
2.8
1.85
37.0 520.0
Recovery
Purification
Elastolytic ActivityC
1.o
26.00
___
0.83 7.80 0.01
Purification based on human protease E esterase activity toward Z-Ala-NP, assayed as described under Methods. b Corrected for nonspecific esterase activity due t o trypsin and chymotrypsin. e Elastolytic activity measured on elastin Congo Red as described under Methods. T h e activity of pure porcine elastase was taken as 100, after 1-hr digest. d Milligrams of 24.5. protein determined using Ezs0 “(,l%) -
trypsin. The significant loss of esterase units was foum !o be primarily due to the removal of large amounts of cationic trypsin and chymotrypsin, both of which show considerable 2-Ala-NP esterase activity (Visser and Blout, 1972; Janoff, 1969). DEAE-SEPHADEXA-50 CHROMATOGRAPHY. The peak tubes of the SE-Sephadex chromatography were pooled and applied to a DEAE-Sephadex A-50 column equilibrated against buffer A. After the passage of inactive unretarded protein, human protease E was eluted by initiation of a linear gradient from 0.025 M CaClz to 0.125 M CaC12, both in 0.005 M Pipes buffer (pH 6.5). As shown in Figure 2, human protease E eluted separately from anionic trypsin a t about 0.1 M CaC12 concentration. The purification scheme is shown in Table 1. Because of the instability of anionic trypsin, any delay following SE-Sephadex chromatography resulted in substantially lower amounts of this enzyme whereas the recovery of human protease E was not noticeably affected. Criteria of Homogeneity and Molecular Weight Studies. Human protease E routinely eluted from DEAE-Sephadex A-50 with constant specific activity. Rechromatography of peak fractions on DEAE-Sephadex resulted in a single peak with constant specific activity identical with the applied material. Additional evidence of the homogeneity of this preparation was obtained by subjecting it to gel filtration on Sephadex (3-75. The preparation eluted as a single symmetrical peak with a constant specific activity comparable to that of the applied enzyme. Analytical disc electrophoresis of human protease E in 7.5% gels a t p H 2.3 and 8.3 showed only a single band with no minor contaminants, again indicative of homogeneity (Figure 3). Sedimentation velocity experiments were performed on iPr2FP enzyme in 0.005 M Pipes-0.05 M CaC12 (pH 6.5) a t protein concentrations of IO and 7.5 mg/ml; both resulted in singly symmetrical Schlieren peaks and a sedimentation constant ( ~ 2 0 . ~of) 3.19 S was calculated from these data. Boundary depletion sedimentation equilibrium experiments were used to calculate the molecular weight of iPr2FP human protease E. A protein concentration of 0.19 mg/ml in 0.005 M Pipes-0.025 M CaC12 (pH 6.5) was used with a rotor speed of 40,000 rpm. The molecular weight calculated from these data was 31,059, using a partial specific volume of 0.722 calculated from the amino acid composiof 24.5 was tion. An extinction coefficient (El cm(l%)) computed from ultracentrifuge studies using interference optics (Babul and Stellwagen, 1969). The extinction coefficient experiments were conducted in 0.005 M Pipes-0,025
FIGURE 3: Polyacryiamme ansc eiectropnoresis ot human protease E (80 pg). Patterns were stained with 1% Amido Schware in 7.5% acetic acid (A) pH 2.3, 7.5% gel, direction of migration is from anode (top) 10 cathode (bottom): ( B ) pH 8.3, 7.5%. direction of migration is from cathode (tap) to anode (bottom).
C a C h (pH 6.5) using iPr2FP enzyme concentrations of 1.7 and 2.7 mg/ml. Sodium dodecyl sulfate gel electrophoresis of iPr2FP human protease E was performed after incubation in sodium dodecyl sulfate solution in order to determine both homogeneity and molecular weight. A single component wa* detected and a molecular weight of 30,500 was estimated using proteins of known molecular weights as standards. The molecular weight of human protease E as estimated by gel filtration chromatography on Sephadex G-100 was 30.000. Amino Acid Composition. The amino acid composition of human protease E is presented in Table 11. These data were calculated from analysis after 22-, 48-, and 72-hr hydrolysis of I-mg samples of enzyme. The number of residues of each amino acid present was based on a n assumed value of 20 residues of alanine per molecule of protein. A molecular weight of 30,792 was calculated from these data. The amino acid compositions of porcine elastase (Shotton, 1970) and the human “elastase 1” isolated by Fzinstein et al. (1974) are also presented in Table I1 for comparison. Stability. Human protease E was quite stable in dilute solutions of protein concentrations less than 0.4 mg/ml within a pH range of 4.0-8.0. No loss of activity could be M
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__ __-_
_..
___.___-__ ~
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TRAVIS
-.
-_ -
Table 11: Amino Acid Compositiona of Human Protease E and Other Related Proteases. Human Protease E
Other Related P r o t e a s e s ____
Time of Hydrolysis -
Amino Acid
22 h r
48 hr
72 hi-
Value Taken
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half -Cystine \'aline Me thi iiriiiie Isoleucine Leucine Tyrosine Phenylalanine Tryptophan
8.2 6.7 10.9 28.5 16.1 23.9 24.2 18.3 31.3
9 .o 8.1 9.6 27.4 15.4 23.8 25.1 15.2 35.9
11.I 7.4 10.0 29.4 14.9 23.6 24.1 15.8 38.4
22.1
22.9
24.9
12.4 20.1 8.5 7.6
12.9 20.6 7.6 7.1
13.3 20.9 7.3 6.4
9.4 7.4 10.0 28.4 16.4' 24.Zb 24.4 17.4 35.2 20.0 15.3' 25.3' 0 .9' 13.7' 20.5 7 .a 7.1 9 .Od N o . of residues Mol wt
Near e s t Integer 9 7 10 28 16 24 24 17 35 20 15 25
1 14 21
8 7 9 290 30.792
P o r c me Elastase'
Human "E last as e l>>f
3 8 6 6 12 10 24 25 19 14 22 16 19 21 7 16 25 26 17 16 8 16 27 24 2 0 10 10 18 17 11 7 3 6 7 ____ -1.1 .d 240 238 25.900 25.050
________ _ - __ . . ~ Data are expressed as amino acid residues/molecule, assuming 20 alanines/molecule of protein. b Threonine and serine values are based on linear extrapolations to zero time, and valine and isoleucine values are based on linear extrapolation t o 96 hr. Averages of 22-hr hydrolysates of oxidized samples. Tryptophan determined by average of 22-hr hydrolysates in the presence of 4 % thioglycolate. e Shotton (1970). f Feinstein e t al. (1974). -
~
a
Table 111: Inhibition of' Human Protease E" with Active-Site Specific Peptide Chloromethyl Ketone Inhibitors of Porcine Pancreatic Elastase. - __~ _ _ _ _ ~ ~ ~ _ _ _ _ _ _ . ._ ~ _ _ _ _ Relative Reactivity'
_
Inhibitor 0.7 7 1.o 1.o Ac-Ala-Ala-AlaCH,Cl 6.5 10 7.5 75 10.7 1.6 Z -Gly - Leu-AlaCH,Cl 6.5 10 20.4 204 34.6 18 Ac - Ala- Ala- Ala- AlaC H,C 1 6.5 10 6.5 10 61 610 88 71 Ac -Ala- Ala- P r o - AlaCH,Cl 13.8 138 19.7 16 Ac -Ala- Ala- Phe - AlaCH,C 1 6.5 10 _______ __ .a Human protease E concentration: 10-5 M, all enzyme assays were performed using t-Boc-Ala-NP as described under Methods. Kinetic constants calculated by the method of Powers and Tuhy (1973). Averages of at least four runs. Controls containing no inhibitor exhibited no significant decrease in esterolytic activity. Relative reactivity assuming Ac-Ala-AlaAlaCH2C1 is equal to 1.0. These values for relative comparison only (see Discussion). These constants were taken from Powers and Tuhy (1973) and were calculated by Powers and Tuhy (1973) for p H 6.5 from original inhibition studies a t p H 5.0. ~
detected after 3 months, even at 24'. Below p H 4.0, however, the enzyme gradually lost activity, presumably due to irreversible denaturation. At protein concentrations above 0.4 mg/ml, rapid autolysis occurred; therefore, for experiments requiring high protein concentrations, iPrzFP enzyme was employed. The pH optimum for protease E was determined on casein substrates using the following buffers: 0.1 M sodium citrate (pH 2.0-5.5), 0.1 M Pipes-HCI (pH 6.0-7.7), 0.1 21 Tris-HCI (pH 7.2-8.0), and 0.1 M sodium carbonate (pH 7.7- 10.5). The pH optimum for casein hydrolysis was be-
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tween pH 7.7 and p H 9.5. Effect of Inhibitors on Human Protease E. S Y ~ T I H I ; . T I < ' I N H I B I T O R S . In order to determine the degree of similarity between porcine elastase and human protease E, several synthetic active-site directed reagents designed specifically to inhibit porcine elastase were tested (Powers and Tuhy. 1973). The data were processed using a least-squarcs computer program and the results are listed in Table 111. All of the compounds tested were found to be strong inhibitors of human protease E a t p H 6.5. NATURALLYO C C U R R I N G INHIBITORS. Human prOte-
_
HUMAN
PANCREATIC ENZYMES,
PROTEASE E
Table IV: Inhibition Spectrum of Human Protease Ea (Expressed as % Proteolytic Activity Remaining).
Table V: Relative Proteolytic Activitya of Several Human and Porcine Pancreatic Proteases. Relative Activity
Molar Ratio Inhibitor : Enzyme Porcine Inhibitor Soybean trypsin Lima bean trypsin Kunitz bovine pancreatic trypsin Chicken ovomucoid porcine pancreatic secretory trypsin Bowman-Birk (soybean) Human s e r u m a,-antitrypsin
1:l 83 96 60 100 95 96 0
5:l 64 91 55 96 91 94
1O:l 59 85 39 89 85 86
Assays for inhibition of proteolytic activity were performed at p H 8.0 as described under Methods. a
ase E was also tested against a wide range of naturally occurring inhibitors including soybean trypsin inhibitor, lima bean trypsin inhibitor, Kunitz bovine pancreatic trypsin inhibitor, chicken ovomucoid, porcine Kazal inhibitor, Bowman-Birk (soybean) trypsin inhibitor, and a-l-antitrypsin. With the notable exception of a-1-antitrypsin, all the naturally occurring inhibitors tested showed a surprising lack of inhibition even at inhibitor-enzyme molar ratios of 1O:l. These results are summarized in Table IV. Although detailed results have not been published, porcine elastase has reportedly little if any susceptibility to chicken ovomucoid (Mandl, 1962), pancreatic trypsin inhibitor (Mandl, 1962), or the Bowman-Birk soybean inhibitor (Gertier and Birk, 1970). Human serum a t 1:lOO dilutions, however, has been found to inhibit porcine elastase from 50 to 90%, while human protease E (0.1 mg/ml) is totally inhibited by a 1: 100 dilution of human serum. In particular, human protease E and porcine pancreatic elastase have both been shown to be subject to complete inhibition by a-1-antitrypsin even a t 1: 1 molar ratios of inhibitor to enzyme. Substrate Specificity and Kinetic Parameters. Human protease E was tested against three synthetic substrates which are commonly used to measure elastase activity. The human enzyme was found to have strong activity toward Z-Ala-NP and t-Boc-Ala-NP. The specific activity of human protease E toward the former was 25.8 as compared to 26.9 for porcine elastase. The K, value for the latter substrate using human protease E was determined from eight different substrate concentrations (ranging from 2.0Km to O.lK,)) to be 1.66 X M. The K , value for this substrate using porcine elastase was reported by Visser and Blout (1972) to be 3.0 X l o w 4 M . Ac(Ala)3OMe, a highly specific substrate for porcine elastase (Gertler and Hofmann, 1970), was also hydrolyzed by human protease E but a t only 25% the rate of that for porcine elastase. Human protease E was also tested against Suc(Ala)3NA, another highly specific substrate for porcine elastase (Bieth et al., 1974). Although the substrate was hydrolyzed, the rate was only 7.5% of that of porcine elastase. This was, however, 500 times greater than the rate of substrate hydrolysis by porcine chymotrypsin and 1000 times greater than hydrolysis by porcine trypsin (Bieth et al., 1974). Human protease E showed no detectable activity toward BzArgOEt or TosArgOMe, both synthetic substrates for trypsin or toward AcTyrOEt or BzLeuOEt which a r e used to detect human and bovine chymotrypsin and bovine chy-
Protein Substrate
Elas-
Casein Undyed elastinb Elastin-Orceinb Elastin Congo Red
100 100
tase
100 100
Human Porcine Protease Trypsin E 98 1.50 0.66 0.17
0.50 0.28 0.10
Human Trypsin 108
0.70 0.04 0.06
a Porcine elastase has been assigned a value of 100. Assay procedure as described by Shotton (1970). Values obtained from 8-hr incubation of protease with substrate.
motrypsin C activity, respectively. In addition, no carboxypeptidase A or B activity could be measured using hippurylL-phenylalanine and hippuryl-L-arginine as substrates. Strong proteolytic activity against casein and hemoglobin was observed with human protease E representing 98% of that shown by porcine elastase when equal milligram quantities of both proteases were tested on these same substrates. Assays f o r Elastolytic Activity. Using the procedure as described by Shotton (1 970), Congo Red-elastin, orceinelastin, and powdered bovine elastin were used to assay for elastolytic activity. Although a strong elastolytic activity was detected in the initial steps of the purification procedure, the protein which we have isolated has no significant elastolytic activity. In addition to the above procedures, the elastin-plate assay as described by Sbarra et a f . (1960) also yielded negative results. The adsorption of human protease E on elastin was measured by the method of Gertler (1971). Using an assay mixture containing 0.125 mg of human protease E in 4.0 ml of 0.1 M borate buffer (pH 8.8), we found that less than 1% of the C A P esterase activity of human protease E was adsorbed on 20 mg of elastin. In identical experiments with porcine elastase, 96% of the Z-Ala-NP esterase activity was adsorbed on elastin and complete digestion occurred within 2 hr. In order to ascertain the absolute elastolytic activity of human protease E as related to other pancreatic proteases, equal microgram quantities of porcine elastase, porcine trypsin, human protease E, and human trypsin were tested against a variety of elastin substrates. The results of this experiment are shown in Table V. Although human protease E does demonstrate trace activity against the elastin substrates tested, this activity would appear to be insufficient to justify calling this enzyme an elastase. Discussion Previous communications regarding the properties of human pancreatic proteolytic enzymes have suggested that there are two elastases present in activated extracts of human pancreatic tissue and in activated human pancreatic juice (Feinstein et al., 1974; Clemente et al., 1972). In our laboratory, however, we have been able to detect only one protein component which possesses elastolytic activity. Yet, we have resolved two proteases which show strong elastaselike esterase activity. The more anionic of these components we have called human protease E. Although this enzyme does have many properties in common with the well-characBIOCHEMISTRY, VOL.
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Table VI: Comparative Properties of Human Protease E and Porcine Elastase. ~~~___________
Property
Human Protease E
Porcine Elastase@
3.19 S
2.60 S
31,059 30,792
25,000 25,900
Extinction coefficient
24.5
23.6
( E j c m(lrY>l) pH optimum I
